Chromosomal Expression of Foreign and Native Genes From Regulatable Promoters in Escherichia coli

A two-step cloning system for expression of foreign and native genes from heterologous promoters in single copy from the E. coli chromosome is described. The system is based on the conditional-replication integration and modular CRIM plasmid technology and new CRIM plasmids described herein. The gene of interest is first synthesized by Polymerase chain reaction (PCR) by using primers with specially designed Sap I site extensions and cloned into the Sap I CRIM cloning plasmid pKZ20. The gene is then subcloned with Sap I into a CRIM expression plasmid, such as pKZ14, pLZ41, or pLZ42, which carry the regulatable promoter araBp8, rhaBp3 , or lac UV5 , respectively. The system is described for gfp , which encodes green fluorescence protein, as an example. The resulting CRIM expression plasmids are then integrated in single copy into a chromosomal phage attachment site by supplying integrase from a helper plasmid. Such integrants can be used for conditional expression of any target gene in single copy on the chromosome, especially in gene-structure-function studies where it is important to avoid copy-number artifacts.